1.1 Field of the Invention PA0 1.2 Cancer PA0 1.3. Cancer Genes PA0 1.3.1. Oncogenes PA0 1.3.2. Tumor Suppressor Genes PA0 1.3.2.1. The Retinoblastoma Gene PA0 1.3.2.2. The Neurofibromatosis Gene PA0 1.3.3.3. The p53 Gene PA0 1.3.3.4. The Deleted in Colon Carcinoma Gene (DCC) PA0 1.4 Genetic Therapy: Gene Transfer Methods PA0 1.5 Proposed Strategies for Cancer Gene Therapy PA0 1.6 Tumor Suppressor Gene Resistance PA0 1.7 Summary of Obstacles to Cancer Gene Therapy
This invention is in the field of tumor suppressor genes (anti-oncogenes) and relates in general to products and methods for practicing broad-spectrum tumor suppressor gene therapy of various human cancers. In particulars the invention relates to methods for treating tumor cells (1) administering vectors comprising a nucleic acid sequence coding for a second in-frame AUG codon-initiated retinoblastoma protein of about 94 kD or (2) administering an effective amount of a protein coded for by the nucleic acid sequence.
Cancers and tumors are the second most prevalent cause of death in the United States, causing 450,000 deaths per year. One in three Americans will develop cancer, and one in five will die of cancer (Scientific American Medicine, part 12, I, 1, section dated 1987). While substantial progress has been made in identifying some of the likely environmental and hereditary causes of cancer, the statistics for the cancer death rate indicates a need for substantial improvement in the therapy for cancer and related diseases and disorders
A number of so-called cancer genes, i.e., genes that have been implicated in the etiology of cancer, have been identified in connection with hereditary forms of cancer and in a large number of well-studied tumor cells. Study of cancer genes has helped provide some understanding of the process of tumorigenesis. While a great deal more remains to be learned about cancer genes, the presently known cancer genes serve as useful models for understanding tumorigenesis.
Cancer genes are broadly classified into "oncogenes" which, when activated, promote tumorigenesis, and "tumor suppressor genes" which, when damaged, fail to suppress tumorigenesis. While these classifications provide a useful method for conceptualizing tumorigenesis, it is also possible that a particular gene may play differing roles depending upon the particular allelic form of that gene, its regulatory elements, the genetic background and the tissue environment in which it is operating.
The oncogenes are somatic cell genes that are mutated from their wild-type alleles (the art refers to these wild-type alleles as protooncogenes) into forms which are able to induce tumorigenesis under certain conditions. There is presently a substantial literature on known and putative oncogenes and the various alleles of these oncogenes. In order to provide background information and to further the understanding of the scope of the invention, a brief discussion of representative oncogenes is provided.
For example, the oncogenes ras and myc are considered as models for understanding oncogenic processes in general The ras oncogene is believed to encode a cytoplasmic protein, and the myc oncogene is believed to encode a nuclear protein. Neither the ras oncogene nor the myc oncogene alone is able to induce full transformation of a normal cell into a tumor cell, but full tumorigenesis usually occurs when both the ras and myc oncogenes are present and expressed together in the same cell (Weinberg, R. A., 1989, Cancer Research 49:3713-3721, at page 3713). Such collaborative effects have been observed between a number of other studied oncogenes.
The collaborative model of oncogene tumorigenesis must be qualified by the observation that a cell expressing the ras oncogene that is surrounded by normal cells does not undergo full transformation. However, if most of the surrounding cells are also ras-expressing, then the ras oncogene alone is sufficient to induce tumorigenesis in a ras-expressing cell. This observation validates the multiple hit theory of tumorigenesis because a change in the tissue environment of the cell hosting the oncogene may be considered a second hit.
An alternative and equally valid hypothesis is that events that collaborate with the activation of an oncogene such as ras or myc may include the inactivation of a negative regulatory factor or factors (Weinberg, R. A., 1989, Cancer Research 49:3713-3721, at 3717; Goodrich, D. W. and Lee, W-H., 1992, Nature 360:177-179), i.e., a tumor suppressor protein.
Tumor suppressor genes are genes that, in their wild-type alleles, express proteins that suppress abnormal cellular proliferation. When the gene coding for a tumor suppressor protein is mutated or deleted, the resulting mutant protein or the complete lack of tumor suppressor protein expression may fail to correctly regulate cellular proliferation, and abnormal cellular proliferation may take place, particularly if there is already existing damage to the cellular regulatory mechanism A number of well-studied human tumors and tumor cell lines have been shown to have missing or nonfunctional tumor suppressor genes. Examples of tumor suppression genes include, but are not limited to, the retinoblastoma susceptibility gene or RB gene, the p53 gene, the deleted in colon carcinoma (DCC) gene and the neurofibromatosis type 1 (NF-1) tumor suppressor gene (Weinberg, R. A. Science, 1991, 254:1138-1146). Loss of function or inactivation of tumor suppressor genes may play a central role in the initiation and/or progression of a significant number of human cancers.
The list of putative tumor suppressor genes is large and growing. The following discussion of tumor suppressor genes is not intended to provide a complete review of all known and putative tumor suppressor genes, but is provided as background to indicate the state of the art and the problems to be overcome before the art is able to provide successful genetic therapy of diseases and disorders characterized by abnormally proliferating cells, e.g., tumor or cancer cells.
The RB gene is one of the better studied tumor suppressor genes. The size of the RB gene complementary DNA (cDNA), about 4.7 Kb, permits ready manipulation of the gene, so that insertions of the RB gene have been made into a number of cell lines. The RB gene has been shown to be missing or defective in a majority of retinoblastomas, sarcomas of the soft tissues and bones, and in approximately 20 to 40 percent of breast, lung, prostate and bladder carcinomas (Lee, W-H., et al., PCT Publ. No. WO 90/05180, at pages 38 and 39; see also, Bookstein, R. and Lee, W-H., 1991, Crit. Rev. Oncog., 2:211-217; Benedict, W. F. et al., J. Clin. Invests., 1990, 85:988-993).
Based upon study of the isolated RB cDNA clone, the predicted RB gene product has 928 amino acids and an expected molecular weight of 106 kD (Lee et al., 1987, Nature, 329:642-645). The natural factor corresponding to the predicted RB gene expression product has been identified as a nuclear phosphoprotein having an apparent relative molecular mass (Mr) of 110-114 kD (Lee et al., 1987, Nature, 329:642-645) or 110-116 kD (Xu et al., 1989, Oncogene 4:807-812). Hence, the literature generally refers to the protein encoded by the RB gene as p110.sup.RB. In this connection, it is noteworthy that measurement of apparent relative molecular mass by SDS-PAGE is frequently inaccurate owing to protein secondary structure Therefore, the full length RB protein of 928 amino acids is also referred to as the 115 kD (Yokota et ale, 1988, Oncogene, 3:471-475), or 105 kD (Whyte et al., 1988, Nature, 334:124-129) RB proteins Various mutations of the RB gene are known. These are generally inactive However, a 56 kD truncated RB protein, designated as p56.sup.RB, that is considered to function in the same way as does p110.sup.RB retains activity (Goodrich et al., 1992, Nature 360:177-179).
on SDS-PAGE normal human cells show an RB protein pattern consisting of a lower sharp band with an Mr of 110 kD and a broader, more variable region above this band with an Mr ranging from 110 kD to 116 kD. The 110 kD band is the under phosphorylated RB protein, whereas the broader region represents the phosphorylated RB protein. The heterogeneity of the molecular mass results from a varying degree of phosphorylation (Xu et al., 1989, Oncogene, 4:807-812).
The RB protein shows cyclical changes in phosphorylation. Host RB protein is unphosphorylated during G1 phase, but most (perhaps all) RB molecules are phosphorylated in S and G2 phases (Xu et al., 1989, Oncogene, 4:807-812; DeCaprio et al., 1989, Cell, 58:1085-1095; Buchkovich et al., 1989, Cell, 58:1097-1105; Chen et al., 1989, Cell, 58:1193-1198; Mihara et al., 1989, Science, 246:1300-1303). Furthermore, only the under phosphorylated RB protein binds to SV40 large T antigen. Given that RB protein binding by large T antigen is probably important for the growth promoting effects of large T antigen, this suggests that the under phosphorylated RB protein is the active form of the RB protein, and the phosphorylated RB protein in S and G2 phases is inactive (Ludlow et al., 1989, Cell, 56:57-65).
The RB gene expressing the first in-frame AUG codon-initiated RB protein is also referred to herein as the intact RB gene, the RB.sup.110 gene or the p110.sup.RB coding gene. It has also been observed that lower molecular weight (&lt;100 kD, 98 kD, or 98-104 kD) bands of unknown origin which are immunoreactive to various anti-RB antibodies can be detected in immunoprecipitation and Western blots (Xu et al., 1989, Oncogene, 4:807-812; Furukawa et al., 1990, Proc. Natl. Acad. Sci., USA, 87:2770-2774; Stein et al., 1990, Science, 249,666-669).
Considering that the RB.sup.110 cDNA open reading frame sequence (McGee, T. L., et al., 1989, Gene, 80:119-128) reveals an in-frame second AUG codon located at exon 3, nucleotides 355-357, the deduced second AUG codon-initiated RB protein would be 98 kD, or 12 kD smaller than the p110.sup.RB protein. It has been proposed that the lower molecular weight bands are the under phosphorylated (98 kD) and phosphorylated (98-104 kD) RB protein translated from the second AUG codon of the RB mRNA (Xu et al., 1989, Oncogene, 4:807-812), although no data directly supported this hypothesis. Thus, no conclusive observation confirms the actual expression of the RB gene from the second in-frame AUG codon. Further, Sections 4.2.1, and FIG. 5 infra provide data indicating the non-identity of the 98 kD protein bands of unknown origin and the second AUG codoninitiated protein products
It has been proposed that introduction of a functional RB.sup.110 gene into an RB-minus tumor cell will likely "normalize" the cell of course, it is not expected that tumor cells which already have normal RB.sup.110 gene expression ("RB+") will respond to RB.sup.110 gene therapy, because it is presumed that adding additional RB expression cannot correct a non-RB genetic defect In fact, it has been shown that in the case of RB+ tumor cell lines, such as the osteosarcoma cell line, U-2 OS, which expresses the normal p110.sup.RB, introduction of an extra p110.sup.RB coding gene did not change the neoplastic phenotype of such tumor lines (Huang, et al., 1988, Science, 242:1563-1566).
In the only reported exception, introduction of a p110.sup.RB coding vector into normal human fibroblasts, WS1, which have no known RB or any other genetic defects, led to the cessation of cell growth (WO 91/15580, Research Development Foundation, by Fung et al., PCT application filed Apr. 10, 1991, published Oct. 17, 1991, at page 18). However, it is believed that these findings were misinterpreted since a plasmid, ppVUO-Neo, producing SV40 T antigen with a well-known growth-promoting effect on host cells was used improperly to provide a comparison with the effect of RB.sup.110 expression on cell growth of transfected WS1 fibroblasts (Fung, et al. Id. see Example 2 page 25). This view is confirmed by the extensive literature, together with similar confirming data provided by the examples presented infra, clearly characterizing RB+ tumor cells as "incurable" by treatment with wild-type RB.sup.110 gene. In addition, it is noteworthy that the WS1 cell line per se is a generally recognized non-tumorigenic human diploid fibroblast cell line with limited cell division potential in culture. Therefore, W091/15580 simply does not provide any method for effectively treating RB+ tumors with an RB.sup.110 gene. Thus, there remains a need for a broad-spectrum tumor suppressor gene for treating abnormally proliferating cells having any type of genetic defect.
Neurofibromatosis type 1 or von Recklinghausen neurofibromatosis results from the inheritance of a predisposing mutant allele or from alleles created through new germline mutations (C. J. Marshall, 1991, Cell, 64:313-326). The neurofibromatosis type 1 gene, referred to as the NF1 gene, is a relatively large locus exhibiting a mutation rate of around 10.sup.4. Defects in the NF1 gene result in a spectrum of clinical syndromes ranging from cafe-au-lait spots to neurofibromas of the skin and peripheral nerves to Schwannomas and neurofibrosarcomas.
The NF1 gene encodes a protein of about 2485 amino acids that shares structural similarity with three proteins that interact with the products of the ras protooncogene (Weinberg et al., 1991, Science, 2541138-1146 at page 1141). For example, the NF1 amino acid sequence shows sequence homology to the catalytic domain of ras GAP, a GTPase-activating protein for p21 ras (C. J. Harshall, 1991, Cell, 64:313-326 at pages 320 and 321).
The role of NF1 in cell cycle regulation is apparently a complex one that is not yet fully elucidated. For example, it has been hypothesized that it is a suppressor of oncogenically activated p21 ras in yeast (C. J. Marshall, (1991, Cell, 64:313-326, bridging pages 320 and 321, and citing to Ballester et al, 1990, Cell, 63:851-859). On the other hand, other possible pathways for NF1 interaction are suggested by the available data (C. J. Marshall, 1991, Cell, 64:313-326 at page 321; Weinberg et al., 1991, Science, 254:1138-1146 at page 1141).
At present, no attempts to treat NF1 cells with a wild-type NF1 gene have been undertaken due to the size and complexity of the NF1 locus. Therefore, it would be highly desirable to have a broad-spectrum tumor suppressor gene able to treat NF1 and any other type of cancer or tumor.
Somatic cell mutations of the p53 gene are said to be the most frequently mutated gene in human cancer (Weinberg et al., 1991, Science, 254:1138-1146 at page 1143). The normal or wild-type p53 gene is a negative regulator of cell growth, which, when damaged, favors cell transformation (Weinberg et al. supra). As noted for the RB protein, the p53 expression product is found in the nucleus, where it may act in parallel with or cooperatively with p110.sup.RB. This is suggested by a number of observations, for example, both p53 and p110 .sup.RB proteins are targeted for binding or destruction by the oncoproteins of SV40, adenovirus and human papillomavirus.
Tumor cell lines deleted for p53 have been successfully treated with wild-type p53 vector to reduce tumorigenicity (Baker, S. J., et al., 1990, Science, 249:912-915). However, the introduction of either p53 or RB.sup.110 into cells that have not undergone lesions at these loci does not affect cell proliferation (Marshall, C. J. 1991, Cell, 64:313-326 at page 321; Baker, S. J. et al., 1990, Science, 249:912-915; Huang, H.-J.S., et al., 1988 Science, 242:1563-1566). Such experiments suggest that sensitivity of cells to the suppression of their growth by a tumor suppressor gene is dependent on the genetic alterations that have taken place in the cells. Such a dependency would be further complicated by the observation in certain cancers that alterations in the p53 tumor suppressor or gene locus appear after mutational activation of the ras oncogene (Marshall, C. J., 1991, Cell, 64:313-326; Fearon, E. R., and Vogelstein, B., 1990, Cell, 61:759-767).
Therefore, there remains a need for a broad-spectrum tumor suppressor gene that does not depend on the specific identification of each mutated gene causing abnormal cellular proliferation.
The multiple steps in the tumorigenesis of colon cancer are readily monitored during development by colonoscopy. The combination of colonoscopy with the biopsy of the involved tissue has uncovered a number of degenerative genetic pathways leading to the result of a malignant tumor. One well studied pathway begins with large polyps of which 60% of the cells carry a mutated, activated allele of K-ras. A majority of these tumors then proceed to the inactivation-mutation of the gene referred to as the deleted in colon carcinoma (DCC) gene, followed by the inactivation of the p53 tumor suppressor gene.
The DCC gene is a more than approximately one million base pair gene coding for a 190-kD transmembrane phosphoprotein which is hypothesized to be a receptor (Weinberg et al., 1991, Science, 254:1138-1146 at page 1141), the loss of which allows the affected cell a growth advantage. It has also been noted that the DCC has partial sequence homology to the neural cell adhesion molecule (Marshall, 1991, Cell, 64:313-326) which might suggest a role for the DCC protogene in regulating cell to cell interactions.
As can be appreciated, the large size and complexity of the DCC gene, together with the complexity of the K-ras, p53 and possibly other genes involved in colon cancer tumorigenesis demonstrates a need for a broad-spectrum tumor suppressor gene and methods of treating colon carcinoma cells which do not depend upon manipulation of the DCC gene or on the identification of other specific damaged genes in colon carcinoma cells.
The treatment of human disease by gene transfer has now moved from the theoretical to the practical realm. The first human gene therapy trial was begun in September 1990 and involved transfer of the adenosine deaminase (ADA) gene into lymphocytes of a patient having an otherwise lethal defect in this enzyme, which produces immune deficiency The results of this initial trial have been very encouraging and have helped to stimulate further clinical trials (Culver, K. W., Anderson, W. F., Blaese, R. M., Hum. Gene. Ther., 1991, 2:107).
So far all but one of the approved gene transfer trials in humans rely on retroviral vectors for gene transduction. Retroviral vectors in this context are retroviruses from which all viral genes have been removed or altered so that no viral proteins are made in cells infected with the vector. Viral replication functions are provided by the use of retrovirus `packaging` cells that produce all of the viral proteins but that do not produce infectious virus Introduction of the retroviral vector DNA into packaging cells results in production of virions that carry vector RNA and can infect target cells, but no further virus spread occurs after infection. To distinguish this process from a natural virus infection where the virus continues to replicate and spread, the term transduction rather than infection is often used.
The major advantages of retroviral vectors for gene therapy are the high efficiency of gene transfer into replicating cells, the precise integration of the transferred genes into cellular DNA, and the lack of further spread of the sequences after gene transduction (Miller, A. D. Nature, 1992, 357:455-460).
The potential for production of replication-competent (helper) virus during the production of retroviral vectors remains a concern, although for practical purposes this problem has been solved. So far, all FDA-approved retroviral vectors have been made by using PA317 amphotropic retrovirus packaging cells (Miller, A. D., and Buttimore, C., Molec Cell Biol., 1986, 6:2895-2902). Use of vectors having little or no overlap with viral sequences in the PA317 cells eliminates helper virus production even by stringent assays that allow for amplification of such events (Lynch, C. H., and Miller, A. D., J. Viral., 1991, 65:3887-3890). Other packaging cell lines are available. For example, cell lines designed for separating different retroviral coding regions onto different plasmids should reduce the possibility of helper virus production by recombination. Vectors produced by such packaging cell lines may also provide an efficient system for human gene therapy (Miller, A. D., 1992, Nature, 357:455-460).
Non-retroviral vectors have been considered for use in genetic therapy. One such alternative is the adenovirus (Rosenfeld, M. A., et al., 1992, Cell, 68:143155; Jaffe, H. A. et al., 1992, Nature Genetics 1.372-378; Lemarchand, P. et al., 1992, Proc. Natl. Acad. Sci. USA, 89:6482-6486). Major advantages of adenovirus vectors are their potential to carry large segments of DNA (36 Kb genome), a very high titre (10.sup.11 ml.sup.-1), ability to infect non-replicating cells, and suitability for infecting tissues in situ, especially in the lung. The most striking use of this vector so far is to deliver a human cystic fibrosis transmembrane conductance regulator (CFTR) gene by intratracheal instillation to airway epithelium in cotton rats (Rosenfeld, M. A., et al., Cell, 1992, 63:143-155). Similarly, herpes viruses may also prove valuable for human gene therapy (Wolfe, J. H. et al., 1992, Nature Genetics, 1:379-384). Of course, any other suitable viral vector may be used for genetic therapy with the present invention.
The other gene transfer method that has been approved by the FDA for use in humans is the transfer of plasmid DNA in liposomes directly to human cells in situ (Nabel, E. G., et al., 1990, Science, 249:1285-1288). Plasmid DNA should be easy to certify for use in human gene therapy because, unlike retroviral vectors, it can be purified to homogeneity In addition to liposome-mediated DNA transfer, several other physical DNA transfer methods such as those targeting the DNA to receptors on cells by completing the plasmid DNA to proteins have shown promise in human gene therapy (Wu, G. Y., et al., 1991, J. Biol. Chem., 266:14338-14342; Curiel, D. T., et al., 1991, Proc. Natl. Acad. Sci. USA, 88:8850-8854).
It has been observed that certain tumor cells return to normal function when fused with normal cells, suggesting that replacement of a missing factor, such as a wild-type tumor suppressor gene expression product may serve to restore a tumor cell to a normal state (reviewed by Weinberg, R. A., 1989, Cancer Research 49:3713-3721, at 3717)
These observations have led to research aimed at providing genetic treatment of tumor cells having defective tumor suppressor genes. The proposed method of treatment requires identification of the damaged tumor suppressor gene, and introduction of the corresponding undamaged gene (including a promoter and a complete encoding sequence) into the affected tumor cells by means of a vector such as a retrovirus able to express the gene product It is proposed that the incorporated functional gene will convert the target cell to a non-malignant state.
For example, The Regents of the University of California, in Patent Cooperation Treaty patent application (by Lee et al., number WO 90/05180, having an international filing date of Oct. 30, 1989 and published May 17, 1990), disclose a scheme for identifying an inactive or defective tumor suppressor gene and then replacing such a defective gene with its functional equivalent. In particular, the WO 90/05180 application proposes, based on in vitro studies, to insert a functional RB.sup.110 gene into an RB-minus tumor cell by means of a retroviral vector in order to render such cells non-malignant.
In addition, international application WO 89/06703 (by Dryja et al., having an international filing date of Jan. 23, 1989, and published Jul. 27, 1989) proposes the treatment of retinoblastoma defective tumors by administering a retinoblastoma gene expression product.
In this connection, it has been reported that the introduction of the RB.sup.11O gene into RB-minus retinoblastoma, osteosarcoma, bladder and prostate carcinoma cells resulted in cells showing reduced tumorigenicity in nude mice, but probably not a reduced cell growth rate. The results varied depending on the particular parental cell line (Goodrich et al., 1992, Cancer Research 52:1968-1973; Banerjee, A., et al., 1992, Cancer Research, 52:6297-6304; Takahashi, R., et al., 1991, Proc. Natl. Acad. Sci., USA, 88:5257-5261; Xu, H-J., et al., 1991, Cancer Research, 51:4481-4485; Bookstein et al, 1990, Science, 247:712-715; Huang, H-J. S., et al., 1988, Science 242, 1563-1566). However, the suppression of tumorigenicity by introduction of the p110.sup.RB coding gene into RB-minus tumor cells is incomplete. The p110.sup.RB reconstituted tumor cells still form invasive tumors in nude mice (Xu, H-J , et al., 1991, Cancer Research, 51:4481-4485; Takahashi, R., et al., 1991, Proc. Natl. Acad. Sci., USA, 88:5257-5261; Banerjee, A., et al., 1992, Cancer Research, 52:6297-6304). In particular, it has been shown that p110.sup.RB reconstituted retinoblastoma cells inoculated into an orthotopic site (in this instance, the eye) consistently produced tumors (Xu, H-J., et al., 1991, Cancer Research 51:4481-4485). These findings, which will be discussed in detail infra, caution that the tumor suppressor gene replacement therapy as heretofore envisioned may simply result in cells that only appear to be "cured" Certainly, the findings of Xu et al. indicate a need for an improved genetic therapy for tumors which avoids these shortcomings.
Another proposed method of treating cancer by gene therapy is to antagonize the function of an oncogene by placing an artificial gene, constructed to have an inverted nucleotide sequence compared to the oncogene, into a tumor cell (U.S. Pat. No. 4,740,463, issued Apr. 26, 1988 by Weinberg, et al.).
All of these proposed solutions also share the deficiency of requiring that the specific genetic defect of the tumor to be treated be identified prior to treatment.
Since the p110.sup.RB protein product is active in the under phosphorylated state (discussed in detail supra), and phosphoamino acid analysis has demonstrated only phosphoserine and phosphothreonine but not phosphotyrosine in RB protein (Shew, J-Y, et al., 1989, Ocogene Research, 1:205-213), it has been proposed to make a mutant RB protein with its serine or threonine residues being replaced by alanine or valine or others and that introduction of such a mutant, unphosphorylated RB protein into target cells may lead to growth arrest (International Application WO 91/15580, Research Development Foundation, by Fung et al., at page 20). Unfortunately, in all cases analyzed so far, the human RB protein carrying a point mutation and retaining the unphosphorylated state were invariably inactive proteins and associated with tumorigenesis rather than tumor suppression (Templeton et al., 1991, Proc. Natl. Acad. Sci., USA, 88,3033-3037.
As the above discussion of gene mutations in tumor cells has indicated, not every cancer gene is a suitable candidate for wild-type gene replacement therapy due to the gene size or complexity or for other reasons The retinoblastoma gene is one of those tumor suppressor genes that is readily accessible to study, thus it provides a model for understanding some of the other disadvantages to cancer gene replacement therapy as heretofore understood.
It is known that reintroduction of the retinoblastoma tumor suppressor gene into RB-defective tumor cells inhibits the tumor cell growth and suppresses the neoplastic phenotype of the target cells (WO 90/05180, cited supra; Huang et al, 1988, Science, 242:1563-1566; Bookstein et al, 1990, Science, 247:712-715; Xu et al, 1991, Cancer Res., 51:4481-4485; Takahashi et al., 1991, Proc. Natl. Acad. Sci., USA, 88:5257-5261; Goodrich et al., 1992, Cancer Res., 52:1968-1973; Banerjee et al., 1992, Cancer Res., 52:6297-6304).
However, the suppression of tumorigenicity is often incomplete. A significant percentage of the RB-reconstituted tumor cells still form small tumors after a longer latency period in nude mouse tumorigenicity assays. Such tumors, although retaining normal RB expression, are histologically malignant and invasive (Xu et al., 1991, Cancer Res., 51:4481-4485; Takahashi et al., 1991, Proc. Natl. Acad. Sci., USA, 88:5257-5261; Banerjee et al., 1992, Cancer Res., 52:6297-6304).
Furthermore, it has been observed that several cell lines derived from such RB-positive tumors have become very tumorigenic and have formed large, progressively growing tumors when subsequently injected into nude mice (Zhou, Y.; Li, J.; Xu, K.; Hu, S-X.; Benedict, W. F., and Xu, H-J., Proc. Am. Assoc. Cancer Res., 34:3214, 1993). This phenomenon, which is referred to herein as tumor suppressor gene resistance (TSGR) is a serious obstacle to the successful implementation of any scheme of tumor suppressor gene therapy for human cancers.
Without wishing to be bound by any particular hypothesis or explanation of the TSGR phenomenon, it is believed that the RB gene product exemplifies a possible explanation for TSGR. RB proteins have an active form (under phosphorylated protein) and an inactive form (phosphorylated protein). Therefore, RB-positive tumor cells may have inherited or acquired the ability to phosphorylate RB proteins to the inactive state and allow tumor cell proliferation to continues Thus, conversion of RB-minus cells with plasmid or virus vectors coding for the p110.sup.RB protein provides only incomplete suppression, or even exacerbation of a percentage of the malignant cell population because the p110.sup.RB protein remains phosphorylated and inactive in some of the target cells.
Alternatively, the tumor cells expressing the RB.sup.110 gene may simply have again inactivated the RB.sup.110 gene by mutation in subsequent cell divisions (Lee et al., 1990,, Immunol. Ser. 51.169-200, at page 188). Thus, there remains a need for a method of treating tumor cells by gene therapy so that the possibility of further mutation and resurgence of malignancy is avoided.
In brief, there are at least three major obstacles to be overcome to achieve a practical tumor suppressor gene therapy for tumor cells:
1) The necessity to determine the identity and sequence of each defective tumor suppressor gene or oncogene before attempting genetic therapy of that tumor. This is particularly a problem considering the multiple genetic defects found in many tumor cells studied; PA1 2) The size and complexity of certain tumor suppressor genes or oncogenes renders manipulation of certain of these genes difficult; and PA1 3) The possibility that TSGR as described above for the RB.sup.110 model system will generate tumor cells that have equal or greater dysfunction than did the original abnormal cells.
Accordingly, there is a need in the art for a genetic therapy for tumor or cancer cells which can safely overcome these problems and provide an effective treatment for all types of tumor cells without the need to determine the exact genetic deficiency of each treated tumor cell and without the risk of TSGR resurgence and exacerbation of the malignancy.